Determination of the Mass Transfer Coefficient (kLa)

in Bioreactor
Nursyahirah Binti Mohd Nazir (2021868142)

Abstract - Modern biopharmaceuticals frequently culture cells

in bioreactors. Changes in the culture environment variables,
such as agitation, nutrients, aeration, and pH, have a significant
impact on how sensitive cells are. While the volumetric mass
transfer coefficient (KLa) depends on the constant of a certain
reactor's mechanics. The static or gassing out approach was
employed in the experiment to determine the KLa value of a
stirred tank reactor with bubble aeration. The agitation speed
parameters are 100, 200, 300, and 400 rpm, and the aeration rate
parameters are 0.5, 1.0, 1.5, 2.0, and 2.5 L/min. Meanwhile, six
different temperatures—25, 30, 35, 40, 45, and 50—are used in
the experiment.

Keywords— Bioreactor; temperature; agitation; aeration;

mass transfer coefficient

I. INTRODUCTION Fig. 1. Schematic Diagram of Bioreactor

A bioreactor is a vessel that supports a biologically active
environment where chemical reactions are carried out using The oxygen usually diffuses through the overlay to the cell
living organisms or compounds that are produced by living culture medium in the bioreactor, and the oxygen from the
organisms that are biochemically active. There are two types sparges dissolves in the cell culture through convection with
of this process: aerobic and anaerobic. The oxygen supply to the aid of agitation. The oxygen bubbles are dispersed by
the broths is frequently insufficient to meet the needs of the agitation, which also facilitates mass transfer across the gas-
bacteria in biochemical reactions. Due to the limited solubility liquid interface. The geometrical parameter of the bioreactor is
of oxygen in the medium, oxygen transfer is frequently the the rate of oxygen transfer (OTR) from the gas to liquid
limiting factor in aerobic bioprocesses, making aeration a interface.
crucial component of commercial aerobic fermentations.
However, the experiment is being conducted using the gassing
out approach in the absence of germs.
The oxygen mass transfer in a stirred tank bioreactor depends
on several factors, including the vessel's shape, the liquid's
physical characteristics, and the operational circumstances.
Pharmaceutical, enzyme, food product, and other
manufacturing processes all greatly benefit from the use of
bioreactors [1]. In order to start the reaction and produce the
required product, it is crucial to maintain the proper
concentration of dissolved oxygen. An essential piece of
knowledge regarding a bioprocess or bioreactor is provided by Fig. 2. Phases of Bacterial Growth
the measurement of kLa. These measures make sure that the
processing conditions are such that there is a sufficient supply In a bioreactor, microbial growth takes the form of a growth
of oxygen accessible for the quickly growing cells. curve with lag, exponential, stationary, and, if the culture is
maintained long enough, a death phase. The cell concentration
begins to increase slightly during the lag period. The cells are
creating enzymes, adjusting to their new surroundings, and
preparing to start reproducing. The cell grows at a rate
proportionate to its concentration during the subsequent phase
of exponential growth. All the enzyme's metabolising routes
for the substrate are set up at this point. Cells are therefor
dividing at their greatest rate. The stationary phase follows. The oxygen uptake rate (OUR) is the rate at which bacteria
Since the cells have occupied the smallest amount of or other microorganisms consume oxygen (typical units of
biological space, there is no net growth rate. Lastly, is death mmol O2/L.h)
phase where the live in cell concentration decreasing. OUR = qO2.X
Where,
II. OBJECTIVES
qO2 = specific rate of oxygen consumption (mmol O2/dgw.h)
The purpose of this experiment is to measure the X = bacteria concentration (gdw/L)
volumetric mass transfer coefficient (kLa) of a stirred tank gdw = gram dry weight of cells
reactor with bubble aeration.
Substituting the OUR and OTR equation in above equation.
III. THEORY dCL/dt = kLa (C* - CL) – qO2.X
Most biological reactions need oxygen as a source in order to
The plot of CL against dCL/dt + qO2X, the slope equal to -
produce their desired results. Fermentation is one example. The
1/kLA.
process of creating compounds from substrates while utilising
organisms is known as fermentation. The oxygen supply is
necessary for the reaction to start and produce the desired
product when there are organisms present. So, in every aerobic
fermentation, the quantity of dissolved oxygen becomes a key
control variable. Understanding the oxygen transport to cells in
a reactor in its whole is crucial. As oxygen is consumed by the
organism during an aerobic fermentation, oxygen must be
continuously added to the reaction liquid in order to maintain
the intended concentration.
By mechanically stirring, the oxygen is broken up and
mixed after being transported from air bubbles and then Fig. 3. CL against dCL/dt + qO2X
sparged into the reaction solution. The oxygen mass transfer
capacity of a reactor is influenced by two variables: air flow
rate and degree of agitation. In aerobic bioreactors, these two Static Gassing Out Method
parameters have a large impact on the mass transfer coefficient,
kLa. The mass transfer coefficient, kLa, depends on the The increase in dissolved oxygen concentration is given by the
mechanics of a certain reactor, specifically on its operating following equation.
conditions and constant dimension.
dCL/dt = kLA (C* - CL)
Both of these methods share a similar underlying principle of
Integrating the equation yield to the following equation,
biological aerobic processes and use graphical techniques to
calculate experimental results for kLa. ∫ dCL/(C* - CL) = ∫ kLA dt
dCL/dt = Oxygen Transfer Rate – Oxygen Uptake Rate ln (C* - CL) = kLA t
Where, The plot of the ln (C* - CL) against time, the slope equals to -
kLA.
CL = dissolved oxygen concentration (mmol O2/L or mgO2/L)

Dynamic Gassing Out Method

The oxygen transfer rate, which can be represented in terms of
kLA, is the rate at which oxygen is transferred into solution.
OTR = kLA (C* - CL)
Where,
kL = oxygen transfer coefficient (cm/h) Fig. 4. ln (C* - CL) against time
a = gas-liquid interfacial area (cm2/cm3)
kLa = volumetric oxygen transfer coefficient (h-1)
C* = saturated dissolved oxygen concentration (mg/L) IV. PROCEDURES
CL = actual dissolved oxygen concentration in the broth (mg/L) Effect of Aeration:
OTR = oxygen transfer rate (mg O2/L.h) 1. The apparatus being set up by ensuring well
conditions of the reactor.
 2. pO2 probe was polarized for two hours before the 8. For every five seconds interval, the value of the
main experiment was started. actual dissolved oxygen concentration, pO2 (%) was
3. The agitation parameter was set up at 400 rpm and recorded.
the temperature parameter, 30°C. 9. Steps above was repeated for different temperature
4. The pump was switch off. parameters which are 30, 35, 40, 45 and 50°C.
5. First aeration parameter was set up at 0.5 L/min and
prepared for two-point calibration. V. RESULTS AND DISCUSSIONS
6. Purging of nitrogen gas takes place until the partial
pressure of the oxygen inside the system reached 0%. The saturated oxygen concentration in water (C*) varies with
7. Nitrogen gas tube was then detached from the reactor temperature. Values for C* are given in the table below:
and pump was switched on again to allow aeration
inside the tank. Table 1. Saturated Oxygen Concentration in Water (C*) Varies with
Temperature
8. For every five seconds time interval, the value of the
actual dissolved oxygen concentration, pO2 (%) was Solubility (Air, 1 atm)
Temperature, T (°C)
recorded. mmol O2/L mg O2/L
9. Steps above was repeated for different parameters 0 0.456 14.6
which is 1.0, 1.5, 2.0, and 2.5 L/min.
10 0.355 11.4
Effect of Agitation: 15 0.322 10.3
1. The apparatus was set up by ensuring well conditions 20 0.288 9.24
of the reactor.
25 0.263 8.42
2. pO2 probe was polarized for two hours before main
30 0.242 7.75
experiment was started.
3. Aeration parameter was set up at 2.0 L/min and the 35 0.228 7.29
temperature parameter, 30°C. 40 0.215 6.90
4. The pump was switched off.
5. First agitation parameter was set up at 100 rpm and
prepared for a two point.
6. Purging of nitrogen gas takes place until the partial
pressure of the oxygen inside the system reached 0%.
7. The nitrogen gas tube was then detached from the
reactor and the pump was switched on again to allow
aeration inside the tank.
8. For every five seconds interval, the value of actual
dissolved oxygen concentration, pO2 (%) was
recorded.
9. Steps above was repeated for different agitation
parameters which are 200, 300 and 400 rpm. Fig.5. Effect of different aeration at constant temperature and agitation

Effect of Temperature: The oxygen concentration curve is shown to drastically

1. The apparatus was set up by ensuring well conditions decrease over time in the figure above. The rate of oxygen
of the reactor. mass transfer or volumetric mass transfer coefficient increases
together with the degree of aeration.
2. pO2 probe was polarized for two hours before the
main experiment was started. Table 2. Volumetric Mass Transfer Coefficient at Varying Aeration
3. The aeration parameter was set up at 2.0 L/min and Magnitude
the agitation parameter at 400 rpm. Aeration (L/min) 0.5 1.0 1.5 2.0 2.5
4. The pump was switch off. Volumetric Mass
5. First temperature parameter was set up at 25°C and Transfer Coefficient, 1.0010 0.9935 0.9873 0.9869 0.9766
kLA (s-1)
prepared for a two-point calibration.
6. Purging of nitrogen gas takes place until the partial
pressure of the oxygen inside the system reached 0%.
7. Nitrogen gas tube was then detached from the reactor
and the pump was switched on again to allow
aeration inside the tank.
 Fig. 6. Volumetric mass transfer coefficient at varying aeration Fig.9. Effect of different temperature at constant agitation and aeration

The Ln oxygen concentration curve is shown in the above

figure to dramatically decrease over time. The volumetric
mass transfer coefficient or oxygen mass transfer rate also
increases as the temperature increases.

Table 4. Volumetric Mass Transfer Coefficient at Varying Agitation

Magnitude
Aeration (L/min) 25 30 35 40 45 50
Volumetric
Mass Transfer
1.0090 1.0025 1.0092 1.0024 1.0026 1.0027
Coefficient, kLA
Fig. 7. Effect of different agitation at constant temperature and aeration (s-1)

The ln oxygen concentration curve is shown in the above

figure to dramatically decrease with time. The rate of oxygen
mass transfer or the volumetric mass transfer coefficient
decreases as the intensity of the agitation increases.

Table 3. Volumetric Mass Transfer Coefficient at Varying Agitation

Magnitude
Aeration (L/min) 100 200 300 400
Volumetric Mass Transfer
1.0187 1.0178 1.0010 1.0090
Coefficient, kLA (s-1)

Fig. 10. Volumetric mass transfer coefficient at varying temperature

The slope of the tangent of the driving force versus time

logarithm curve is used to calculate the mass transfer
coefficient. The dissolved oxygen content, which is taken to
be the partial pressure of the gas inside the reactor, is what
drives the reaction. It can be assumed that the system has
reached the maximum saturation point for the oxygen gas
when the partial pressure of oxygen gas reaches a constant
value.

Aeration is the first operating parameter and one of the most

Fig. 8. Volumetric mass transfer coefficient at varying agitation important components of a bioreactor in order to maintain
optimum oxygen availability throughout the system. In order
to eliminate dissolved gaseous, aeration aids in mixing the gas
bubbles through the liquid medium where water and air are
brought into close contact. At aeration, the volumetric mass
transfer coefficient is equivalent to 0.5, 1.0, 1.5, 2.0, and 2.5
litres per minute (1.0010, 0.9935, 0.9873, 0.9868, and 0.9766,
respectively).
The second parameter is agitation. The agitation
processes serve to uniformly distribute gas bubbles in water,
which is necessary for the dispersion of air or oxygen.
Additionally, by forcing the gas bubbles to the tank's bottom
and improving mass transfer, it extends the gas's time of
retention in the water. However, the increased agitation might
result in an increase in power usage. At agitation speeds of
200, 300, and 400 rpm, respectively, the volumetric mass
transfer coefficients are 1.0187, 1.0178, 1.0010, and 1.0090.
Then we have temperature. Both the volumetric mass
transfer coefficient and oxygen solubility in a medium are
adversely impacted by rising temperatures. As the temperature
rises, oxygen solubility decreases in pure water. At
temperatures of 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C, the
volumetric mass transfer coefficient is, accordingly, 1.0090,
1.0025, 1.0092, 1.0024, 1.0026, and 1.0027.
CONCLUSION
The experiment involved examining the three parameters of
temperature, agitation rate, and aeration rate. At 100 rpm, 200
rpm, 300 rpm, and 400 rpm, the volumetric mass transfer
coefficient was 1.0187, 1.0178, 1.0010, and 1.0090,
respectively. The graph created using these data demonstrated
that the kLa value decreased with increasing agitation rate.
The kLa values for the aeration rate were 1.0010, 0.9935,
0.9873, 0.9868, and 0.9766. The volumetric mass transfer
coefficient values at 30 oC are inversely correlated with the
increase in aeration rate. Combining all of these factors, it is
assumed that agitation rate, followed by aeration and
temperature, is the most important factor.
RECOMMENDATIONS
There are a few recommendations for improving the
performance in this experiment. First and foremost, the
gassing out method employs two methods—dynamic and
static—for calculating the mass transfer coefficient, kLa. To
get a good outcome, students can either employ both
approaches or the dynamic method. Then, they can compare
the experimental data produced using the two ways. As soon
as the nitrogen gas is released, students must move on to the
next step of the experiment as quickly as they can to prevent
oxygen from spreading out inside the bioreactor and affecting
the percentage of oxygen concentration that is present inside
the tank. As a result, it's crucial to check that the oxygen level
in the tank has dropped to zero after purging.
REFERENCES
[1] M. Chitra, "Dissolved Oxygen Control of Batch
Bioreactor using Model Reference Adaptive Control
scheme," IFAC-PapersOnLine, 2018.
[2] Y. Zhou, "Effects of Agitation, Aeration and Temperature
on Production of a Novel Glycoprotein GP-1 by
Streptomyces kanasenisi ZX01 and Scale-Up Based on
Volumetric Oxygen Transfer Coefficient," National
Library of Medicine, 2011.
[3] L. sciences, "7 factors that affect oxygen transfer to cells
in bioreactors," cytiva, 2017. [Online]. Available:
https://www.cytivalifesciences.com/en/us/solutions/biopr
ocessing/knowledge-center/7-factors-that-affect-oxygen-
transfer-to-cells-in-bioreactors.
[4] D. Moutadchieva, "Experimental determination of the
volumetric mass transfer coefficient," Researchgate,
2013.
 APPENDIX

SAMPLE CALCULATION

Sample calculation for experimental driving force for O2 concentration:

Sample calculation for slope of the tangent of the ln oxygen concentration curve: